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1 Department of Microbiology and Immunology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-shi, Tokyo 183,
The2 2nd Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
To investigate the viral replication cycle and genomic heterogeneity of hepatitis C virus (HCV), we established an HCV cultivation system by using a primary hepatocyte culture from patients with chronic hepatitis C. Liver tissue was obtained by needle biopsy or surgery, then hepatocytes were isolated by collagenase digestion. After several weeks, we determined the HCV RNA titre of the cultured cells and supernatant by a competitive polymerase chain reaction (PCR) method. A significant amount of HCV RNA was observed in the cells and supernatant during cultivation. Negative-strand RNA, regarded as a marker of viral replication, could be detected by a strand-specific reverse transcription PCR method and the HCV core protein could be detected by immunofluorescence microscopy. Many HCV particles released into the supernatant were infectious. In addition, we compared the nucleotide sequences in the E2/NS1 region of pre- and post-cultivation hepatocytes for 8 weeks. At the beginning of the culture period, three major HCV types containing two subtypes were isolated. Following cultivation, the same types were isolated from the cultured hepatocytes in the same ratio as prior to cultivation. We could detect the same clones in this patient's serum, but in vivo we observed genetic variability over a 6 month interval. One clone detected throughout the 6 month period mutated extensively in the hypervariable region. These results indicated that HCV can replicate in cultured hepatocytes, and that infectious virions are released into the supernatant. This cultivation system should facilitate the study of HCV genomic heterogeneity, infection and replication.
* Author for correspondence. Fax +81 423 21 8678. e-mail yasui@to-shinkeiken.ac.jp
Received 11 September 1995;
accepted 19 December 1995.
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